title: Development and optimization of an internally controlled dried blood spot assay for surveillance of human immunodeficiency virus type-1 drug resistance.
abstract: OBJECTIVE
  We present the evaluation of a methodology for the genotypic assessment of human immunodeficiency virus type-1 (HIV-1) drug resistance, optimized for use with dried blood spots (DBS).
  
  
  METHODS
  The ability to generate HIV-1 protease (PR) and reverse transcriptase (RT) contiguous amplicons and nucleotide sequences from DBS was evaluated. Different collection matrices and extraction methodologies were compared. The relative subtype sensitivity of the amplification strategy was assessed using a comprehensive panel of plasmids representing A-H subtypes. A panel of DBS and plasma specimens was subjected to HIV genotyping. Sequences generated from each sample type were compared.
  
  
  RESULTS
  Extensive replicate testing revealed most sensitivity with the use of 903 filter paper and silica/guanidine extraction, which had an estimated 95% inclusivity endpoint of 1542 proviral copies/mL, as compared with 21 573 proviral copies/mL for the FTA system. All HIV-1 group M subtypes analysed-with the exception of subtypes A2, AE, AG, F and H-had a relative sensitivity of </=10 plasmid copies/PCR reaction. The PCR was multiplexed to include amplification of a human housekeeping gene to monitor the integrity of the human genomic DNA. Using a panel of clinical samples, we demonstrated the ability to amplify and sequence from 83% (n = 10) in the PR region and 100% (n = 12) in the RT region, of samples with detectable viral load. All specimens with an HIV-1 RNA load >/=1000 copies/mL were successfully amplified and sequenced. Twelve specimens had pol genotyping from both plasma and DBS samples. Sequence analysis and drug resistance interpretation revealed that 10 (83%) provided concordant drug resistance interpretation.
  
  
  CONCLUSIONS
  Our results demonstrate that the technique is appropriate for surveillance of drug resistance in untreated individuals and those with virological failure on therapy.
authors.affiliation: Virus Reference Department, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK. andrew.buckton@hpa.org.uk
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Buckton
authors.forename: Sara L
authors.initials: SL
authors.lastname: Bissett
authors.forename: Richard E
authors.initials: RE
authors.lastname: Myers
authors.forename: Simon
authors.initials: S
authors.lastname: Beddows
authors.forename: Simon
authors.initials: S
authors.lastname: Edwards
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Cane
authors.forename: Deenan
authors.initials: D
authors.lastname: Pillay
mesh_terms: D000328:Adult; D019943:Amino Acid Substitution / Q000235:genetics; D001769:Blood / Q000821:virology*; D003890:Desiccation; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000821:virology*; D016333:HIV Protease / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013048:Specimen Handling / Q000379:methods*; D019562:Viral Load
chemical_list: C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1